Here we report the use of structural data from the pre-fusion dengue virus-2 (DENV-2) E protein as a model for a computational approach to the design of new peptide inhibitors of DENV-2 entry. This approach makes use of a residue-specific all-atom probability discriminatory function (RAPDF) score to identify in situ amino acid sequences that are likely to have high structural or and binding stability [23], [24]. Out of seven computationally designed peptides that were synthesized and tested, two were identified as possessing fifty percent in vitro inhibitory activity (IC50) below 10 ��M and another with IC50 activity below 40 ��M. Two of the inhibitors (DN57opt and DN81opt) are binding optimized variants of peptides originally designed from DENV inhibitory peptide sequences located in domain II near the domain I/domain II hinge region [9].

The other (1OAN1) is an entirely novel peptide designed from an extended beta sheet region comprising the first connection between domains I and II. We show that the two peptides with the highest inhibitory activity interfere with virus:cell binding, cause structural changes to the surface of DENV-2 virions, and bind specifically to purified DENV-2 E protein. The causative agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome, DENV has emerged in the past several decades as the most important mosquito borne viral disease with an estimated 2.5 billion people living in areas at risk for epidemic transmission and 50�C100 million people infected annually [25], [26].

Complicating this situation, the four distinct serotypes of DENV generate only low level immunological cross protection, allowing for repeated epidemic outbreaks in the same populations [27], [28]. The phenomenon of antibody dependent enhancement has been shown to result in more severe disease in individuals who have been previously infected with a different serotype [29]�C[33]. With no specific treatment or prevention available other than vector control, DENV is an important target for the development of antivirals and vaccines. The results presented here indicate that the DENV E glycoprotein has multiple accessible surfaces that can be targeted by distinct inhibitors and is an amenable target for rational inhibitor design.

Materials and Methods Computational optimization of hinge region inhibitory peptides Peptide inhibitors were designed to have improved in situ binding compared to naturally occurring sequences using the residue-specific all-atom probability discriminatory function (RAPDF) [24]. The x-ray diffraction structure of DENV-2 envelope protein (Protein Data Bank identifier 1OAN) was used as a template for creating mutant structures from which the peptides were derived [14]. For each peptide, we randomly selected a residue Drug_discovery side chain and substituted it with a new side chain.